PROJECT SUMMARY/ABSTRACT The endosymbiont Wolbachia pipientis is being released into populations of the mosquito vector of dengue, Zika, chikungunya and yellow fever viruses throughout the tropics. In the laboratory, Wolbachia has been shown to ?vaccinate? the vector, reducing or eliminating its ability to transmit viruses. One concern for this biocontrol approach is that the mode of Wolbachia?s action remains unknown. Additionally, as with all interventions (drugs, vaccines and insecticides), the emergence of resistance may threaten the long-term efficacy of this endosymbiont. Here, we use powerful Evolve and Resequence approaches in the mosquito, Wolbachia and in the virus to (1) understand the likely mechanism of pathogen blocking, and (2) determine the most likely genetic paths through which resistance will emerge in the mosquito and the virus. First, we will select for both improved and lessened Wolbachia-mediated blocking of dengue virus in Wolbachia-infected mosquitoes; we have already shown this is possible in a pilot study. In a fully replicated design, we will track the dengue virus load in the mosquito and Wolbachia densities through time. We will perform RNAseq pre- and post-selection and DNA sequencing throughout the regime on the lines as well as on random controls, demonstrating changes in blocking. Via SNP and expression analyses, we will identify key genes associated with the phenotypic shifts, in both symbiont and vector genomes, and develop a putative model for pathogen blocking. In addition, we will have evidence of how it might be possible to improve the strength of dengue blocking and the likely rate and diversity of evolutionary paths toward resistance. Through selection experiments in cell culture, we will determine which aspects of the virus genome can confer protection against the Wolbachia effect. By examining the fitness of the evolved mosquitoes and of the evolved viruses, we will address the question of whether such variants would be competitive and pose a real threat to evolution in natural environments. This work is novel in its application of real-time evolution rather than a ?sit and wait? approach to see how the relationships evolve in field sites. This proactive strategy may help to design targeted strategies that circumvent the most likely forms of resistance and demonstrate a means to improve upon the level of pathogen blocking exhibited by current release strains.